Germination method of cibotium barometz spores and breeding method of cibotium barometz

Abstract

The invention relates to a germination method of cibotium barometz spores and a breeding method of cibotium barometz, and belongs to the technical field of plant culture. The breeding method comprises the following steps: disinfection: disinfecting cibotium barometz spores for 5-10 minutes by using a 10% sodium hypochlorite solution; spore germination, wherein a used germination culture medium comprises a 1 / 4 MS culture medium, 4.5-7 g.L <-1 > agar and 0-30 g.L <-1 > sucrose; sporophyte differentiation: a used sporophyte differentiation culture medium comprises a 1 / 2 MS culture medium, 0 to 1 mg.L <-16 >-BA, 0.5 to 1 mg.L <-12 >, 4-D, 4.5 to 7 g.L <-1 > of agar and 25 to 30 g.L <-1 > of sucrose; sporophyte seedling strengthening: a used sporophyte seedling strengthening culture medium comprises a 1 / 4 MS culture medium, 4.5 g.L <-1 > to 7 g.L <-1 > agar, 30 g.L <-1 > sucrose and 1 g.L <-1 > to 2 g.L <-1 > activated carbon; acclimatization and transplantation: sealing the tissue culture seedlings, gradually releasing the sealing, and then transferring the tissue culture seedlings into a specific mixed matrix for culture to obtain commercial seedlings. According to the method, the spore germination rate can be increased by adopting a specific disinfection procedure, and the effects of more seedlings and good seedlings can be achieved by adopting a specific culture medium and seedling hardening configuration in each stage.

Description

Technical Field

[0001] The invention relates to the technical field of plant cultivation, in particular to a spore germination method of Cibotium cantoniensis and a breeding method of Cibotium cantoniensis. Background Art

[0002] Cibotium barometz (L.) J.Sm. is a plant of the genus Cibotium in the family Pteridaceae. Chinese medicine believes that Cibotium barometz is bitter, sweet and warm in taste, and has the effects of removing rheumatism, nourishing the liver and kidneys, and strengthening the waist and knees. It is often used to treat back pain, wind-cold dampness, and numbness of hands and feet. The wild resources of Cibotium barometz are widely distributed, mainly in subtropical areas such as Yunnan, Guizhou, Guangdong and Guangxi. Affected by the development of industrial development and utilization and the deterioration of the ecological environment, the wild resources of Cibotium barometz are constantly decreasing, and the state has listed it as a second-level protected plant. In order to ensure the sustainable use of Cibotium barometz resources, artificial planting and breeding have become important means.

[0003] Cibotium barometz can be propagated through three methods: sexual reproduction, division and tissue culture. Sexual reproduction takes a long time and is easily restricted by seasons and climate. Although division has the characteristics of high efficiency and consistent genetic traits, it has a low reproduction coefficient and causes great damage to wild resources. Tissue culture technology can use the regenerative ability of cells to cultivate complete plants, with the characteristics of short cycle, high yield and controllability, and can provide high-quality materials to meet the development of the industry.

[0004] In the tissue culture method of Cibotium caninum spores, there are many different opinions on the spore disinfection procedure and culture system. At present, 70% alcohol combined with 0.1% mercuric chloride or 5% sodium hypochlorite is often used to disinfect spores. Some disinfection steps are cumbersome, mercuric chloride contains highly toxic substances, and the germination effect after disinfection is unclear. In addition, previous studies have mostly established tissue rapid propagation systems through sporophyte germination, prothallus proliferation, sporophyte induction, sporophyte proliferation, sporophyte rooting and seedling hardening steps. The time span is long, and multiple culture media need to be replaced, which increases the complexity of the experiment and results in fewer seedlings. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a method for germinating spores of Cibotium barometz and a method for breeding Cibotium barometz, by adopting a specific and simple disinfection procedure with the highest spore germination rate, and by optimizing the culture medium formula and seedling hardening configuration at each stage to achieve the effect of producing more seedlings and better seedlings.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first object of the present invention is to provide a method for germination of Cibotium spores, comprising the following steps:

[0008] Disinfection: Disinfect the spores of Cibotium chinense with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium chinense spores and add sterile water to prepare a suspension containing Cibotium chinense spores;

[0009] Spore germination: inoculating the suspension containing the Cibotium spores into a germination medium for cultivation, so that the Cibotium spores germinate, transform into young prothallus, and then transform into mature prothallus; wherein the germination medium comprises 1 / 4MS medium, 4.5-7g·L-1 agar and 0-30g·L-1 sucrose.

[0010] The germination method of Cibotium spores of the present invention comprises the following steps: using a 10% sodium hypochlorite solution to disinfect Cibotium spores for 5-10 minutes in the disinfection step, and using a 1 / 4MS culture medium, 4.5-7 g·L -1 Agar and 0-30 g·L -1 The germination medium containing sucrose can significantly improve the germination rate of the golden dog spores, and the golden dog spores can be effectively germinated; and the 1 / 4MS medium is used in the germination medium, and the quantity and proportion of inorganic nutrients are appropriate, which are sufficient to meet the nutritional and physiological needs of the golden dog spore germination, and the 1 / 4MS medium has a low inorganic salt concentration, which is conducive to the germination of the golden dog spores; at the same time, the use of the germination medium is also conducive to the effective conversion of the golden dog spores after germination into young prothallus and mature prothallus.

[0011] As a preferred embodiment of the present invention, the germination method of Cibotium spores of the present invention further includes a material screening step before the disinfection step: collecting leaves of the sporangium of Cibotium spores, collecting spores after the sporangium cracks, and screening with 60-mesh and 150-mesh sieves to obtain pure Cibotium spores, which are stored in an environment of 4°C for standby use. Through the above scheme, the sporangium shells and impurities can be screened out to obtain golden pure Cibotium spores, so that the pure Cibotium spores can be used for disinfection and other subsequent steps.

[0012] As a preferred embodiment of the present invention, in the germination method of the spores of Cibotium chinense, in the disinfection step, the spores of Cibotium chinense are first soaked overnight with sterile water, and then the spores of Cibotium chinense are disinfected with a 10% sodium hypochlorite solution. Soaking the spores of Cibotium chinense with sterile water overnight can allow the spores of Cibotium chinense to absorb water, promote metabolism, and increase the germination rate.

[0013] As a preferred embodiment of the present invention, in the germination method of Cibotium spores of the present invention, in the disinfection step, the disinfection time is 5-7 minutes. By using 10% sodium hypochlorite solution to disinfect Cibotium spores for 5-7 minutes, the germination rate of Cibotium spores can reach 100%, and the Cibotium spores can be effectively germinated.

[0014] As a preferred embodiment of the present invention, in the method for germinating the spores of Cibotium caninum of the present invention, the concentration of the suspension containing the spores of Cibotium caninum is 0.3-0.7 mg / mL, more preferably 0.4-0.6 mg / mL, and most preferably 0.5 mg / mL.

[0015] The second object of the present invention is to provide a method for breeding Cibotium chinense, comprising the disinfection and spore germination steps of any of the above-mentioned Cibotium chinense spore germination methods; and further comprising the following steps:

[0016] Sporophyte differentiation: The mature prothallus mass formed after the spore germination step is inoculated into a sporophyte differentiation medium for cultivation, so that the mature prothallus mass is transformed into a sporophyte mass; wherein the sporophyte differentiation medium comprises 1 / 2MS medium, 0-1 mg·L -1 6-BA, 0.5-1mg·L -1 2,4-D, 4.5-7 g L -1 Agar and 25-30 g L -1 sucrose;

[0017] Sporophyte seedling strengthening: the sporophytes in the sporophyte mass are inoculated into the sporophyte seedling strengthening medium for cultivation, so as to strengthen the sporophyte seedlings and obtain tissue culture seedlings; wherein the sporophyte seedling strengthening medium comprises 1 / 4MS medium, 4.5-7g·L -1 Agar, 30 g L -1 Sucrose, 1-2 g·L -1 Activated carbon;

[0018] Seedling hardening and transplanting: The tissue culture seedlings obtained in the spore seedling strengthening step are sealed and then gradually unsealed, and then the tissue culture seedlings are transferred to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation to obtain commercial seedlings.

[0019] The breeding method of Cibotium chinense disclosed by the present invention can improve the germination rate of Cibotium chinense spores by using 10% sodium hypochlorite solution to disinfect Cibotium chinense spores for 5-10 minutes in the disinfection step, so that the Cibotium chinense spores can be effectively germinated; by specifically designing the formula of the germination medium used in the spore germination step, the sporophyte differentiation medium used in the sporophyte differentiation step and the sporophyte seedling strengthening medium used in the sporophyte seedling strengthening step, and by specifically designing the method and the mixed matrix of the seedling hardening and transplanting step, the breeding method of Cibotium chinense disclosed by the present invention can have a high proliferation coefficient and achieve the effect of more seedlings.

[0020] In the breeding method of Cibotium chinense of the present invention, 10% sodium hypochlorite solution is used to disinfect Cibotium chinense spores for 5-10 minutes in the disinfection step, and a 1 / 4MS culture medium containing 4.5-7 g·L -1 Agar and 0-30 g·L -1 The germination medium containing sucrose is used for cultivation, which improves the germination rate of the golden dog spores and enables the golden dog spores to germinate effectively; and the 1 / 4MS medium is used in the germination medium, and the amount and proportion of inorganic nutrients are appropriate, which is sufficient to meet the nutritional and physiological needs of the golden dog spore germination, and the low inorganic salt concentration of the 1 / 4MS medium is conducive to the germination of the golden dog spores; at the same time, the use of the germination medium is also conducive to the effective conversion of the golden dog spores after germination into young prothallus and mature prothallus, and the proliferation coefficient from young prothallus to mature prothallus is high. By using a 1 / 2MS medium containing 0-1mg·L -1 6-BA, 0.5-1mg·L -1 2,4-D, 4.5-7 g·L -1 Agar and 25-30 g L -1 Cultivation in a sucrose sporophyte differentiation medium can induce the transformation of mature prothallus groups into sporophyte groups, and promote the growth of sporophyte plants on the sporophyte groups, the lengthening of roots, the large number of differentiated sporophytes, and the high proliferation coefficient of the transformation from mature prothallus groups to sporophyte groups; and the addition of 6-BA and 2,4-D hormones (plant growth regulators) to the sporophyte differentiation medium can stimulate the growth of sporophyte plants and stimulate some physiological functions. -1 Agar, 30 g L -1 Sucrose and 1-2 g·L -1 The activated carbon spore seedling medium can be used for cultivation, which can promote the plant growth and rooting of tissue culture seedlings. In the seedling hardening and transplanting step, the tissue culture seedlings after being hardened are first sealed and then gradually unsealed, and then transferred to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation, which can improve the survival rate of tissue culture seedlings and transform tissue culture seedlings into qualified commercial seedlings. Through the coordinated cooperation of the above steps, the breeding method of Cibotium barometz of the present invention can achieve the effect of more seedlings and better seedlings.

[0021] As a preferred embodiment of the present invention, in the breeding method of Cibotium chinense of the present invention, in the spore germination step, the germination medium comprises 1 / 4MS medium, 7g·L -1 Agar and 30 g L -1 Sucrose; in the sporophyte differentiation step, the sporophyte differentiation medium includes 1 / 2MS medium, 0.5 mg·L -16-BA, 1mg·L -1 2,4-D, 7g·L -1 Agar and 30 g L -1 Sucrose; in the spore seedling strengthening step, the spore seedling strengthening medium includes 1 / 4MS medium, 7g·L -1 Agar, 30 g L -1 Sucrose, 1-g·L -1 The present invention adopts a specific disinfection procedure in the disinfection step and a specific 1 / 4MS culture medium, 7g·L -1 Agar and 30 g L -1 Sucrose germination medium, a specific 1 / 2MS medium, 0.5 mg·L -1 6-BA, 1mg·L -1 2,4-D, 7g·L -1 Agar and 30 g L -1 The sporophyte differentiation medium of sucrose is a specific medium including 1 / 4MS medium, 7g·L -1 Agar, 30 g L -1 Sucrose, 1-g·L -1 The activated carbon spore seedling medium is combined with a specific treatment method and a specific mixed matrix in the seedling hardening and transplanting steps, so that the breeding method of Cibotium barometz of the present invention can achieve the effect of more seedlings.

[0022] As a preferred embodiment of the present invention, in the spore germination step, the suspension containing the golden dog spores is inoculated into the germination medium, and then cultured for 21-30 days to germinate the golden dog spores; the culture is continued for 60 days to transform the germinated golden dog spores into young prothallus; the young prothallus is transferred to the new germination medium and cultured for 30 days to transform the young prothallus into mature prothallus; in the sporophyte differentiation step, the mature prothallus mass formed after spore germination is inoculated into the sporophyte differentiation culture, and then cultured for 6 days. 0d, the mature prothallus mass is transformed into a sporophyte mass; in the sporophyte seedling strengthening step, the sporophyte in the sporophyte mass is inoculated into the sporophyte seedling strengthening medium, and then cultured for 30-60d to strengthen the sporophyte seedlings and obtain tissue culture seedlings; in the seedling hardening and transplanting step, the tissue culture seedlings obtained after the sporophyte seedling strengthening step are sealed in the tissue culture bottle for 3d, and the degree of unscrewing the bottle cap is gradually increased on the 4th to 6th day, and then the tissue culture seedlings are transferred to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for culture to obtain commercial seedlings. The breeding method of Cibotium barometz of the present invention has more seedlings.

[0023] As a preferred solution of the present invention, in the seedling hardening and transplanting step, the tissue culture seedlings obtained after the spore seedling hardening step are sealed in the tissue culture bottle under the seedling hardening conditions for 3 days, and the bottle cap is unscrewed 1 / 4 on the 4th day, the bottle cap is unscrewed 1 / 2 on the 5th day, and the bottle cap is fully opened on the 6th day. On the 7th day, the tissue culture seedlings are taken out of the tissue culture bottle and transplanted to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation. The above scheme is used to first perform a sealing treatment on the tissue culture seedlings after the seedling hardening and then gradually release the sealing, and then the tissue culture seedlings are transplanted to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation, which can improve the survival rate of the tissue culture seedlings.

[0024] As a preferred solution of the present invention, in the seedling hardening and transplanting step, after the tissue culture seedlings are taken out from the tissue culture bottle, the culture medium at the roots of the tissue culture seedlings is rinsed clean, 40%-50% of the leaves above the base of the tissue culture seedlings are removed, and then they are transplanted into a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1.

[0025] As a preferred embodiment of the present invention, in the breeding method of Cibotium barometz of the present invention, in the spore germination step, the culture is carried out under the conditions of an ambient temperature of 25±3°C and a fluorescent lamp irradiating for 16 hours per day; in the sporophyte differentiation step, the culture is carried out under the conditions of an ambient temperature of 25±3°C and a fluorescent lamp irradiating for 16 hours per day; in the sporophyte seedling strengthening step, the culture is carried out under the conditions of an ambient temperature of 25±3°C and a fluorescent lamp irradiating for 16 hours per day; in the seedling hardening and transplanting step, the tissue culture seedlings are sealed under the seedling hardening conditions of natural scattered light; after the tissue culture seedlings are transplanted, they are cultured under the conditions of natural scattered light. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings.

[0027] Figure 1 A flow chart of the breeding method of Cibotium chinense of the present invention;

[0028] Figure 2 The germination of the spores of Cibotium chinense in Example 3, Example 1, and Example 2 (from left to right) are shown;

[0029] Figure 3 The germination of the spores of Cibotium chinense of Comparative Example 1, Comparative Example 2 and Comparative Example 3 (from left to right);

[0030] Figure 4 This is a diagram of the sporophyte differentiation of Example 4;

[0031] Figure 5 The figure is a diagram of the sporophyte seedling growth of Example 4;

[0032] Figure 6It is the seedling hardening and transplanting situation diagram of Example 4. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer; the raw materials, reagents, etc. used, unless otherwise specified, are all raw materials and reagents that can be obtained from commercial channels such as conventional markets. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of protection claimed by the present invention.

[0034] 1 Materials

[0035] Select healthy and disease-free Cibotium chinense plants, and pick leaves with brown, plump, and unopened sporangium on the back. Spread the picked leaves flat on newspapers and place them in a cool and ventilated place until the sporangium cracks. Collect the spores scattered on the newspapers, and use 60-mesh and 150-mesh sieves to screen the collected spores, remove the sporangium shells and impurities, and leave the pure golden spore powder. Store the spore powder at 4°C for later use.

[0036] 2. Reagents and instruments

[0037] Reagents: sodium hypochlorite, agar, sucrose, anhydrous ethanol (Tianjin Damao Chemical Reagent Factory), 1 / 4MS medium, 1 / 2MS medium, potato medium (Haibo Biotechnology Co., Ltd.), 6-BA, 2,4-D, HCl, NaOH, activated carbon (Guangzhou Chemical Reagent Factory). Please refer to Tables 1 and 2 for the specific names and formulas of some reagents.

[0038] Table 1 Chinese and English comparison table of reagents

[0039]

[0040] Table 2 MS medium formula

[0041]

[0042]

[0043] Note: The content of potassium nitrate, ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate and calcium chloride in 1 / 2MS and 1 / 4MS culture media is halved respectively, while the rest remain unchanged.

[0044] Instruments and equipment: high pressure sterilizer (Shanghai Boneng Instrument Co., Ltd., CT-90A), clean bench (Sujing Group Sujing Antai Air Technology Co., Ltd., VS-1300L-U), microscope (Olybas Bioscience, CX43), ultrapure water meter (Xiamen Kexi Instrument Co., Ltd., UPF-40L-RO), pH meter (Shanghai Sanxin Instrument Factory, PHB-3), electronic balance (German Sartorius Group, BS110S), etc.

[0045] 3. Method of the present invention

[0046] 3.1 Germination method of Cibotium caninum spores of the present invention

[0047] The present invention provides a method for germinating spores of Cibotium caninum, comprising the following steps:

[0048] Disinfection: Disinfect the spores of Cibotium chinense with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium chinense spores and add sterile water to prepare a suspension containing Cibotium chinense spores;

[0049] Spore germination: inoculating the suspension containing the golden dog spores into a germination medium for culturing, so that the golden dog spores germinate, transform into young prothallus, and then transform into mature prothallus; wherein the germination medium comprises 1 / 4MS medium, 4.5-7g·L -1 Agar and 0-30 g·L -1 sucrose.

[0050] Example 1

[0051] This embodiment provides a method for germination of Cibotium caninum spores, comprising the following steps:

[0052] (1) Disinfection: Disinfect the spores of Cibotium chinense with 10% sodium hypochlorite solution for 5 minutes; collect the disinfected spores of Cibotium chinense and add sterile water to prepare a suspension containing spores of Cibotium chinense.

[0053] Specifically: weigh 10 mg of golden dog spores, put them into a 2 mL centrifuge tube, add 1 ml of sterile water and soak overnight. The supernatant was sucked off, and 1.5 mL of 10% sodium hypochlorite solution was added for disinfection for 5 min. During the 5 min disinfection process, the centrifuge tube was shaken from time to time to make the golden dog spores and the sodium hypochlorite solution evenly contacted, and finally the centrifuge tube was allowed to stand in advance (within the disinfection time of 5 min, 1.5-2 min in advance) to allow the golden dog spores to precipitate naturally. After the disinfection is completed (i.e., after the golden dog spores are precipitated), the disinfectant was sucked off, and washed with sterile water 4 times to obtain the golden dog spore precipitation. The golden dog spore precipitation was added with 1 mL of sterile water and transferred to a tissue culture bottle, and 19 mL of sterile water was continued to be added to dilute it into a golden dog spore suspension with a concentration of 0.5 mg / mL, and it was set aside.

[0054] (2) Spore germination: inoculating the suspension containing the spores of Cibotium chinense into a tissue culture bottle containing a germination medium to allow the spores of Cibotium chinense to germinate; wherein the germination medium comprises 1 / 4MS medium, 7 g·L -1 Agar and 30 g L -1 sucrose.

[0055] Specifically: prepare germination medium (1 / 4MS medium + 7g·L -1 Agar + 30 g L -1 Sucrose, pH5.8), put the germination medium into the tissue culture bottle, and sterilize it at 121℃ for 20min, then cool it to solidify and set it aside. Use a pipette to draw 0.5mL of the 0.5mg / mL suspension containing Cibotium spores treated as above, and drop it (inoculate) into the germination medium of the tissue culture bottle (the weight of spores in each bottle is about 0.25mg), shake it well to make the Cibotium spores evenly distributed, inoculate 8 bottles, and repeat 3 times. Place the tissue culture bottle in the culture room for culture, and culture it under fluorescent lamps, 25±3℃, and 16h of light per day for 30 days to allow the spores to germinate. (Observe and record the growth status every day after inoculation, and calculate the contamination rate, germination rate and earliest germination time after 30 days.)

[0056] After 30 days of cultivation, the culture was continued for 60 days to allow the germinated spores to transform into young prothallus.

[0057] Then the young prothallus in each tissue culture bottle is divided into 8 bottles, and each bottle is inoculated with 5 clusters of young prothallus with a diameter of 1 cm. This is repeated 3 times. At this time, the weight of the protospores contained in each cluster of young prothallus is 5-6.25 μg. The culture is continued for 30 days to make the diameter of the prothallus about 2.5 cm, that is, the young prothallus is transformed into mature prothallus.

[0058] Example 2

[0059] This embodiment provides a method for germination of Cibotium caninum spores, which is basically the same as that of Embodiment 1, except that the disinfection time of this embodiment is 7 minutes.

[0060] Example 3

[0061] This embodiment provides a method for germination of Cibotium caninum spores, which is basically the same as that of Embodiment 1, except that the disinfection time of this embodiment is 10 minutes.

[0062] Comparative Example 1

[0063] This comparative example provides a method for germination of Cibotium caninum spores, which is basically the same as Example 1, except that: this comparative example uses 4% sodium hypochlorite solution for disinfection for 3 minutes.

[0064] Comparative Example 2

[0065] This comparative example provides a method for germination of Cibotium caninum spores, which is basically the same as Example 1, except that this comparative example uses 4% sodium hypochlorite solution for disinfection for 5 minutes.

[0066] Comparative Example 3

[0067] This comparative example provides a method for germination of Cibotium caninum spores, which is basically the same as Example 1, except that: this comparative example uses 4% sodium hypochlorite solution for disinfection for 7 minutes.

[0068] 3.2 The breeding method of Cibotium chinense of the present invention

[0069] The present invention provides a breeding method for Cibotium chinense. Figure 1 , including the following steps:

[0070] Disinfection: Disinfect the spores of Cibotium chinense with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium chinense spores and add sterile water to prepare a suspension containing Cibotium chinense spores;

[0071] Spore germination: inoculating the suspension containing the golden dog spores into a germination medium for culturing, so that the golden dog spores germinate, transform into young prothallus, and then transform into mature prothallus; wherein the germination medium comprises 1 / 4MS medium, 4.5-7g·L -1 Agar and 0-30 g·L -1 sucrose;

[0072] Sporophyte differentiation: The mature prothallus mass formed after the spore germination step is inoculated into a sporophyte differentiation medium for cultivation, so that the mature prothallus mass is transformed into a sporophyte mass; wherein the sporophyte differentiation medium comprises 1 / 2MS medium, 0-1 mg·L -1 6-BA, 0.5-1mg·L -1 2,4-D, 4.5-7 g·L -1 Agar and 25-30 g L -1 sucrose;

[0073] Sporophyte seedling strengthening: the sporophytes in the sporophyte mass are inoculated into the sporophyte seedling strengthening medium for cultivation, so as to strengthen the sporophyte seedlings and obtain tissue culture seedlings; wherein the sporophyte seedling strengthening medium comprises 1 / 4MS medium, 4.5-7g·L -1 Agar, 30 g L -1 Sucrose, 1-2 g·L -1 Activated carbon;

[0074] Seedling hardening and transplanting: After the spore seedling strengthening step, the tissue culture seedlings are sealed and then gradually unsealed. Then the tissue culture seedlings are transferred to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation to obtain commercial seedlings.

[0075] Example 4

[0076] This embodiment provides a breeding method for Cibotium chinense. Figure 1 , comprising the following steps (wherein the disinfection and germination steps are the same as in Example 1):

[0077] (1) Disinfection: Disinfect the spores of Cibotium chinense with 10% sodium hypochlorite solution for 5 minutes; collect the disinfected spores of Cibotium chinense and add sterile water to prepare a suspension containing spores of Cibotium chinense.

[0078] Specifically: weigh 10 mg of golden dog spores, put them into a 2 mL centrifuge tube, add 1 ml of sterile water and soak overnight. The supernatant was sucked off, and 1.5 mL of 10% sodium hypochlorite solution was added for disinfection for 5 min. During the 5 min disinfection process, the centrifuge tube was shaken from time to time to make the golden dog spores and the sodium hypochlorite solution evenly contacted, and finally the centrifuge tube was allowed to stand in advance (within the disinfection time of 5 min, 1.5-2 min in advance) to allow the golden dog spores to precipitate naturally. After the disinfection is completed (i.e., after the golden dog spores are precipitated), the disinfectant was sucked off, and washed with sterile water 4 times to obtain the golden dog spore precipitation. The golden dog spore precipitation was added with 1 mL of sterile water and transferred to a tissue culture bottle, and 19 mL of sterile water was continued to be added to dilute it into a golden dog spore suspension with a concentration of 0.5 mg / mL, and it was set aside.

[0079] (2) Spore germination: inoculating the suspension containing the spores of Cibotium chinense into a tissue culture bottle containing a germination medium for culturing, so that the spores of Cibotium chinense germinate, transform into young prothallus, and transform into mature prothallus; wherein the germination medium comprises 1 / 4MS medium, 7 g·L -1 Agar and 30 g L -1 sucrose.

[0080] Specifically: prepare germination medium (1 / 4MS medium + 7g·L -1 Agar + 30 g L -1 Sucrose, pH 5.8), put the germination medium into the tissue culture bottle, and sterilize it at 121℃ for 20min, then cool it to solidify and set aside. Use a pipette to draw 0.5mL of the 0.5mg / mL suspension containing golden dog spores treated above, and drop (inoculate) it into the germination medium of the tissue culture bottle (the weight of spores in each bottle is about 0.25mg), shake it well to make the golden dog spores evenly distributed, inoculate 8 bottles, and repeat 3 times. Place the tissue culture bottle in the culture room for 30 days under fluorescent lamps, 25±3℃, and 16h of light per day to allow the spores to germinate.

[0081] After 30 days of cultivation, the culture was continued for 60 days to allow the germinated spores to transform into young prothallus.

[0082] Then the young prothallus in each tissue culture bottle is divided into 8 bottles, and each bottle is inoculated with 5 clusters of young prothallus with a diameter of 1 cm. This is repeated 3 times. At this time, the weight of the protospores contained in each cluster of young prothallus is 5-6.25 μg. The culture is continued for 30 days to make the diameter of the prothallus about 2.5 cm, that is, the young prothallus is transformed into mature prothallus.

[0083] (3) Sporophyte differentiation: The mature prothallus mass formed after the spore germination step is inoculated into a tissue culture bottle containing a sporophyte differentiation medium to transform the mature prothallus mass into a sporophyte mass; wherein the sporophyte differentiation medium comprises 1 / 2MS medium, 0.5 mg·L -1 6-BA, 1mg·L -1 2,4-D, 7g·L -1 Agar, 30 g L -1 sucrose.

[0084] Specifically: prepare sporophyte differentiation medium (1 / 2MS medium + 0.5mg·L -1 6-BA+1mg·L -1 2,4-D+7g·L -1 Agar + 30 g L -1 Sucrose, pH 5.8), the sporophyte differentiation medium is placed in the tissue culture bottle, and sterilized at 121°C for 20 min, then cooled to solidify for later use. The prothallus mass of the mature prothallus formed in step (2) is cut into prothallus masses with a diameter of about 1 cm (the weight of the protospores contained in each prothallus mass is about 1-1.25 μg) and inoculated into the sporophyte differentiation medium of the tissue culture bottle, 5 prothallus masses are inoculated per bottle, 8 bottles are inoculated each time, and repeated 3 times, each bottle is 1 replicate. The tissue culture bottle is placed in the culture room and cultured for 60 days, and the culture conditions are the same as those for spore germination.

[0085] (4) Sporophyte seedling strengthening: The sporophytes of the sporophyte mass after the sporophyte differentiation step are inoculated into a tissue culture bottle containing a sporophyte seedling strengthening medium to strengthen the sporophyte seedlings and obtain tissue culture seedlings; the sporophyte seedling strengthening medium includes 1 / 4MS medium, 7g·L -1 Agar, 30 g L -1 Sucrose, 1g·L -1 Activated carbon.

[0086] Specifically: prepare the spore seedling medium (1 / 4MS medium + 7g·L -1 Agar + 30 g L -1 Sucrose + 1g·L -1Activated carbon, pH 5.8), put the sporophyte seedling medium into the tissue culture bottle, and sterilize it at 121℃ for 20min, then cool it to solidify for later use. Separate the single sporophyte from the sporophyte mass and peel off the prothallus at the bottom, then inoculate it on the sporophyte seedling medium in the tissue culture bottle and culture it for 30d. Inoculate 8 bottles each time, each bottle is a repeat, repeat 3 times, and inoculate 5 sporophytes in each bottle.

[0087] (5) Seedling hardening and transplanting: After the tissue culture seedlings obtained after the spore seedling hardening step are sealed in the tissue culture bottle for 3 days, the degree of unscrewing the bottle cap is gradually increased from the 4th to the 6th day, and the tissue culture seedlings are transferred from the tissue culture bottle to a mixed matrix of peat soil: perlite: coconut bran = 3:1:1 for cultivation to obtain commercial seedlings.

[0088] Specifically: the tissue culture seedlings obtained by cultivation are used as transplanting materials. The tissue culture seedlings to be transplanted are sealed in bottles for 3 days, and on the 4th day, 1 / 4 of the bottle cap is loosened and sprayed with tap water. On the 5th day, 1 / 2 of the bottle cap is opened and sprayed with tap water. On the 7th day, the tissue culture seedlings are clamped out of the tissue culture bottle with tweezers, and the culture medium at the roots is washed off in running water. The washed tissue culture seedlings are temporarily placed in a basin of water, and 40%-50% of the leaves above the base are removed, and then transplanted one by one into a hole tray containing a mixed matrix (mass ratio of charcoal soil: perlite: coconut bran = 3:1:1). After being thoroughly watered with tap water, the lid of the tray is covered to keep moisture. Place it under natural scattered light for 30 days to obtain commercial seedlings.

[0089] Example 5

[0090] This embodiment provides a breeding method for Cibotium barometz, which is basically the same as that of Embodiment 4, except that the disinfection time in this embodiment is 7 minutes.

[0091] Example 6

[0092] This embodiment provides a breeding method for Cibotium barometz, which is basically the same as that of Embodiment 4, except that the disinfection time in this embodiment is 10 minutes.

[0093] Example 7

[0094] This embodiment provides a breeding method for Cibotium barometz, which is basically the same as that of Embodiment 4, except that: the germination medium of this embodiment includes 1 / 4MS medium, 4.5 g·L -1 Agar and 0 g·L -1 Sucrose (i.e., sucrose-free).

[0095] Example 8

[0096] This embodiment provides a breeding method for Cibotium barometz, which is basically the same as that of Embodiment 4, except that: the germination medium of this embodiment includes 1 / 4MS medium, 4.5 g·L-1 Agar and 25 g L -1 sucrose.

[0097] Example 9

[0098] This embodiment provides a breeding method for Cibotium caninum, which is basically the same as that of Embodiment 4, except that: the sporophyte differentiation medium of this embodiment includes 1 / 2MS medium, 1 mg·L -1 6-BA, 0.5mg·L -1 2,4-D, 4.5 g·L -1 Agar and 30 g L -1 sucrose.

[0099] Example 10

[0100] This embodiment provides a breeding method for Cibotium chinense, which is basically the same as that of Embodiment 4, except that: the sporophyte differentiation medium of this embodiment includes 1 / 2MS medium, 0 mg·L -1 6-BA (i.e. 6-BA-free), 1 mg·L -1 2,4-D, 7g·L -1 Agar and 25 g L -1 sucrose.

[0101] Embodiment 11

[0102] This embodiment provides a breeding method for Cibotium chinense, which is basically the same as that of Embodiment 4, except that: the spore seedling medium of this embodiment includes 1 / 4MS medium, 4.5g·L -1 Agar, 30 g L -1 Sucrose, 2 g·L -1 Activated carbon.

[0103] Example 12

[0104] The present embodiment provides a breeding method of Cibotium barometz, which is basically the same as that of Embodiment 4, except that in the sporophyte seedling strengthening step of the present embodiment, the sporophyte is cultured in a sporophyte seedling strengthening medium for 60 days to obtain tissue culture seedlings.

[0105] 4 Results

[0106] 4.1 Germination results of Cibotium spores

[0107] The contamination rate, germination rate and earliest germination time of Examples 1-3 and Comparative Examples 1-3 were respectively counted. Please refer to Table 3 for the results.

[0108] Any abnormal tissue culture phenomenon in the culture medium is considered as contamination.

[0109] Germination rate: number of germination bottles / number of inoculation bottles × 100%;

[0110] Earliest germination time: The germination time is confirmed by observing the appearance of small green spots on the culture medium with the naked eye.

[0111] Table 3 Germination results of Cibotium spores

[0112] Disinfection method Contamination rate (%) Germination rate (%) Earliest germination time (d) Example 1 10% sodium hypochlorite 5min 0 100 21 Example 2 10% sodium hypochlorite 7min 0 100 23 Example 3 10% sodium hypochlorite 10min 0 86.66 26 Comparative Example 1 4% sodium hypochlorite 3min 0 72.22 29 Comparative Example 2 4% sodium hypochlorite 5min 0 83.33 29 Comparative Example 3 4% sodium hypochlorite 7min 0 94.44 28

[0113] As shown in Table 3, the present invention can significantly improve the germination rate of Cibotium spores by using 10% sodium hypochlorite to disinfect Cibotium spores for 5-10 minutes, so that Cibotium spores can germinate effectively and at an early time. Preferably, referring to Example 1 and Example 2, the present invention can achieve a germination rate of 100% of Cibotium spores by using 10% sodium hypochlorite to disinfect Cibotium spores for 5-7 minutes, and the earliest germination time is significantly earlier than the earliest germination time of Comparative Examples 1-3.

[0114] 4.2 Breeding results of Cibotium chinense

[0115] The germination rate, earliest germination time, prothallus proliferation coefficient, differentiation reproduction coefficient, total proliferation coefficient, and total proliferation coefficient variance of Examples 4-12 were statistically analyzed. Please refer to Table 4 for the results.

[0116] Germination rate: number of germination bottles / number of inoculation bottles × 100%;

[0117] Earliest germination time: The earliest germination time is confirmed by observing the appearance of small green spots on the culture medium with the naked eye;

[0118] Prothallus multiplication coefficient: refers to the multiplication coefficient of the transformation from young prothallus to mature prothallus during the spore germination step;

[0119] Differentiation reproduction coefficient: refers to the proliferation coefficient of the sporophyte differentiated from the mature prothallus in the sporophyte differentiation step;

[0120] Total proliferation coefficient: refers to the product of the prothallus proliferation coefficient and the differentiation reproduction coefficient;

[0121] Survival rate of tissue culture seedlings: refers to the number of surviving tissue culture seedlings / total number of transplanted tissue culture seedlings during the seedling hardening and transplanting steps.

[0122] Table 4 Proliferation coefficient results

[0123]

[0124] In the breeding method of Cibotium cinereae of Examples 4-12 of the present invention, in the disinfection and spore germination steps, the contamination rate of Cibotium cinereae spores is 0; the germination rates of Cibotium cinereae spores of Examples 4-5 and 7-12 are all 100%, and the germination rate of Cibotium cinereae spores of Example 6 is 86.66%, which is lower than that of Examples 4-5 and 7-12, but still has a high germination effect; in the breeding method of Cibotium cinereae of Examples 4-12, in the disinfection and spore germination steps, green germinated materials can be observed with the naked eye on the 21st to 26th day of culture, and in the process of continuing to culture the germinated Cibotium cinereae spores to transform into young prothallus and then into mature prothallus, the prothallus proliferation coefficient from young prothallus to mature prothallus is 5.13-5.73. In the sporophyte differentiation step, in the process of culturing the differentiation medium for 60 days to differentiate the mature prothallus into the sporophyte, the differentiation proliferation coefficient of the mature prothallus to differentiate into the sporophyte is 7.13-8.00, and in this process, the sporophyte plant grows taller and the root length increases. In the sporophyte seedling strengthening step, the tissue culture seedling grows taller and the number of roots increases; the proliferation coefficient of the entire culture cycle is 39.43-44.03. In the seedling hardening and transplanting step, by first sealing the tissue culture seedlings and then gradually unscrewing the bottle cap and then transferring them to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation, the survival rate of the tissue culture seedlings can reach 92%, which is significantly improved compared with the survival rate of the tissue culture seedlings that are transplanted without sealing treatment (83%) and the survival rate of the tissue culture seedlings that are transplanted immediately after sealing without gradually opening the cap (83%). Therefore, the breeding method of Cibotium barometz of the present invention has more seedlings.

[0125] Comprehensively considering all the properties, the breeding method of Cibotium chinense in Example 4 has the best breeding effect and the most seedlings. In the breeding method of Cibotium chinense in Example 4, the Cibotium chinense spores are disinfected for 5 minutes by using 10% sodium hypochlorite solution in the disinfection step, and the spore germination step is carried out by using 1 / 4MS medium, 7g·L -1 Agar and 30 g L -1 The germination medium composed of sucrose can make the germination rate of Cibotium spores reach 100%, and Cibotium spores begin to germinate as early as the 21st day of culture. Figure 2 , the germination effect of Example 4 (i.e. corresponding to Figure 2 The embodiment 1) in the above is good. By continuing to culture in the above germination medium, the germinated Cibotium spores are transformed into young prothallus, and the young prothallus are further transformed into mature prothallus. The prothallus proliferation coefficient of the young prothallus transformed into mature prothallus in embodiment 4 is the highest. The breeding method of Cibotium spp. in embodiment 4 adopts a 1 / 2MS medium, 0.5 mg·L -1 6-BA, 1mg·L -1 2,4-D, 7g·L -1Agar and 30 g L -1 The sporophyte differentiation medium containing sucrose was used to culture the mature prothallus mass to transform into the sporophyte mass. The differentiation and proliferation coefficient of the mature prothallus mass to the sporophyte mass in Example 1 was the highest. In addition, the sporophyte plant was also helped to grow taller and the roots longer in the process. For the sporophyte differentiation, please refer to Figure 4 In the method for breeding Cibotium chinense of Example 4, in the step of strengthening the spore seedlings, 1 / 4MS medium and 7 g·L -1 Agar, 30 g L -1 Sucrose, 1-g·L -1 Cultivating with activated carbon spore seedling medium can make the plant grow taller and increase the number of roots. For spore seedling growth, please refer to Figure 5 In the breeding method of Cibotium barometz in Example 4, in the seedling hardening and transplanting step, the tissue culture seedlings are first sealed and then gradually unscrewed and then transferred to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation, so that the survival rate of the tissue culture seedlings can reach 92%. For the hardening and transplanting conditions, please refer to Figure 6 Therefore, the breeding method of Cibotium chinense of Example 4 has the most seedlings, which is achieved by adopting the disinfection procedure with the highest spore germination rate and optimizing the culture medium formula and seedling hardening configuration at each stage to achieve the effect of more and better seedlings.

[0126] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A method for germinating spores of Cibotium caninum, characterized in that: The following steps are involved: Disinfection: Disinfect the spores of Cibotium chinense with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium chinense spores and add sterile water to prepare a suspension containing Cibotium chinense spores; Spore germination: inoculating the suspension containing the golden dog spores into a germination medium for culturing, so that the golden dog spores germinate, transform into young prothallus, and then transform into mature prothallus; wherein the germination medium comprises 1 / 4MS medium, 4.5-7g·L -1 Agar and 0-30 g·L -1 sucrose.

2. The method for germination of Cibotium caninum spores according to claim 1, characterized in that: Before the disinfection step, the method further comprises a material screening step: collecting leaves of the sporangium of the Cibotium chinense plant, collecting spores after the sporangiums are cracked, and screening them with 60-mesh and 150-mesh sieves in succession to obtain pure Cibotium chinense spores.

3. The method for germination of spores of Cibotium caninum according to claim 1, characterized in that: In the disinfection step, the spores of Cibotium caninum are first soaked in sterile water overnight, and then disinfected with 10% sodium hypochlorite solution.

4. The method for germination of spores of Cibotium caninum according to claim 1, characterized in that: In the disinfection step, the disinfection time is 5-7 minutes.

5. The method for germination of spores of Cibotium caninum according to claim 1, characterized in that: In the disinfection step, the concentration of the suspension containing Cibotium caninum spores is 0.3-0.7 mg / mL.

6. The method for germination of spores of Cibotium caninum according to claim 5, characterized in that: In the disinfection step, the concentration of the suspension containing Cibotium caninum spores is 0.4-0.6 mg / mL.

7. The method for germination of spores of Cibotium caninum according to claim 6, characterized in that: In the disinfection step, the concentration of the suspension containing Cibotium caninum spores is 0.5 mg / mL.

8. A method for breeding Cibotium barbatum, characterized in that: The method comprises the disinfection and spore germination steps in the spore germination method of Cibotium caninum according to any one of claims 1 to 7; and further comprises the following steps: Sporophyte differentiation: The mature prothallus mass formed after the spore germination step is inoculated into a sporophyte differentiation medium for cultivation, so that the mature prothallus mass is transformed into a sporophyte mass; wherein the sporophyte differentiation medium comprises 1 / 2MS medium, 0-1 mg·L -1 6-BA, 0.5-1mg·L -1 2,4-D, 4.5-7 g·L -1 Agar and 25-30 g L -1 sucrose; Sporophyte seedling strengthening: the sporophytes in the sporophyte mass are inoculated into the sporophyte seedling strengthening medium for cultivation, so as to strengthen the sporophyte seedlings and obtain tissue culture seedlings; wherein the sporophyte seedling strengthening medium comprises 1 / 4MS medium, 4.5-7g·L -1 Agar, 30 g L -1 Sucrose, 1-2 g·L -1 Activated carbon; Seedling hardening and transplanting: The tissue culture seedlings obtained after the spore seedling hardening step are sealed and then gradually unsealed, and then the tissue culture seedlings are transferred to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation to obtain commercial seedlings.

9. The method for breeding Cibotium barbatum according to claim 8, characterized in that: In the spore germination step, the germination medium includes 1 / 4MS medium, 7g·L -1 Agar and 30 g L -1 sucrose; In the sporophyte differentiation step, the sporophyte differentiation medium includes 1 / 2MS medium, 0.5 mg·L -1 6-BA, 1mg·L -1 2,4-D, 7g·L -1 Agar and 30 g L -1 sucrose; In the spore seedling strengthening step, the spore seedling strengthening culture medium includes 1 / 4MS culture medium, 7g·L -1 Agar, 30 g L -1 Sucrose, 1g·L -1 Activated carbon.

10. The method for breeding Cibotium barbatum according to claim 8, characterized in that: In the spore germination step, the suspension containing the golden dog spores is inoculated into a germination medium, and then cultured for 21-30 days to allow the golden dog spores to germinate; the culture is continued for 60 days to allow the germinated golden dog spores to transform into young prothallus; the young prothallus is transferred to a new germination medium and cultured for another 30 days to allow the young prothallus to transform into mature prothallus; In the sporophyte differentiation step, the mature prothallus mass formed after spore germination is inoculated into the sporophyte differentiation culture and cultured for 60 days to transform the mature prothallus mass into the sporophyte mass; In the step of strengthening the spore seedlings, the spores in the spore mass are inoculated into the strengthening medium and cultured for 30-60 days to strengthen the spore seedlings and obtain tissue culture seedlings; In the seedling hardening and transplanting step, the tissue culture seedlings obtained after the spore seedling hardening step are sealed in the tissue culture bottle for 3 days, and then the degree of unscrewing the bottle cap is gradually increased from the 4th to the 6th day, and then the tissue culture seedlings are transferred to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation to obtain commercial seedlings.

11. The method for breeding Cibotium barbatum according to claim 10, characterized in that: In the seedling hardening and transplanting step, the tissue culture seedlings obtained after the spore seedling strengthening step are sealed in a tissue culture bottle under the seedling hardening condition for 3 days, and then the bottle cap is unscrewed by 1 / 4 on the 4th day, the bottle cap is unscrewed by 1 / 2 on the 5th day, and the bottle cap is fully opened on the 6th day. The tissue culture seedlings are taken out from the tissue culture bottle on the 7th day and transplanted to a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:1 for cultivation.

12. The method for breeding Cibotium barbatum according to claim 11, characterized in that: In the seedling hardening and transplanting step, after the tissue culture seedlings are taken out from the tissue culture bottle, the culture medium at the roots of the tissue culture seedlings is rinsed clean, 40%-50% of the leaves above the base of the tissue culture seedlings are removed, and then they are transplanted into a mixed matrix with a mass ratio of peat soil: perlite: coconut bran = 3:1:

1.

13. The method for breeding Cibotium barometz according to claim 11, characterized in that: In the spore germination step, the sporophyte differentiation step, and the sporophyte seedling strengthening step, the cultivation is carried out under the conditions of an ambient temperature of 25±3°C and 16 hours of fluorescent light per day; in the seedling hardening and transplanting step, the seedling hardening condition is natural diffuse light, and after transplanting, the cultivation is carried out under the condition of natural diffuse light.

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